In-vitro biological evaluation of 3,3′,5,5′-tetramethoxy-biphenyl-4,4′-diol and molecular docking studies on trypanothione reductase and Gp63 from Leishmania amazonensis demonstrated anti-leishmania potential

Available treatments for leishmaniasis have been widely used since the 1940s but come at a high cost, variable efficacy, high toxicity, and adverse side-effects. 3,3′,5,5′-Tetramethoxy-biphenyl-4,4′-diol (TMBP) was synthesized through laccase-catalysis of 2,6-dimethoxyphenol and displayed antioxidant and anticancer activity, and is considered a potential drug candidate. Thus, this study aimed to evaluate the anti-leishmanial effect of TMBP against promastigote and amastigote forms of Leishmania (L.) amazonensis and investigated the mechanisms involved in parasite death. TMBP treatment inhibited the proliferation (IC50 0.62–0.86 µM) and induced the death of promastigote forms by generating reactive oxygen species and mitochondrial dysfunction. In intracellular amastigotes, TMBP reduced the percentage of infected macrophages, being 62.7 times more selective to the parasite (CC50 53.93 µM). TMBP did not hemolyze sheep erythrocytes; indicative of low cytotoxicity. Additionally, molecular docking analysis on two enzyme targets of L. amazonensis: trypanothione reductase (TR) and leishmanolysin (Gp63), suggested that the hydroxyl group could be a pharmacophoric group due to its binding affinity by hydrogen bonds with residues at the active site of both enzymes. TMBP was more selective to the Gp63 target than TR. This is the first report that TMBP is a promising compound to act as an anti-leishmanial agent.

In general, compounds with IC 50 ≤ 10.00 µM are reported in the literature to have potential pharmacologic activity 31 . In this respect, TMBP showed potential antileishmanial activity by completely inhibiting the viability of the parasites, even in the shortest incubation time (24 h), and exhibiting low IC 50 values, ranging from 0.48 to 0.86 μM.
As cell shrinkage is a clear indication of cell death, primarily via apoptotic mechanisms [32][33][34] , we performed a flow cytometry analysis to confirm the antileishmanial effect of TMBP. The shortest period (24 h) was chosen for this set of experiments since there was no difference between the IC 50  www.nature.com/scientificreports/   www.nature.com/scientificreports/ indicated a reduction in the cell size of the promastigotes treated with TMBP at concentrations ranging from 1.95 to 15.6 μM for 24 h (Fig. 3), suggesting the loss of cell volume, which is the predominant characteristic of apoptosis 33 . Apoptosis-related death has also been reported for other phenolic compounds, including gallic acid, quercetin, and rutin, through increasing nitric oxide production and causing apoptosis and DNA damage, which presented structural changes in cells that led to the death of L. donovani 35 .
On confirming the anti-promastigote effect of TMBP, we investigated the mechanisms of action involved in parasitic death by evaluating the production of reactive oxygen species (ROS) and the mitochondrial integrity in the parasites.
Due to their unique mitochondria, the Leishmania genus parasites require the maintenance of mitochondrial integrity for survival 36 . Studies have demonstrated that changes in mitochondrial integrity caused by drugs, such as antimony and sterol methenyl transferase (SMT) inhibitors, are related to cell death of L. amazonensis 37 .
The TMRE probe was used to assess the possible induction of alterations in the integrity of the mitochondrial potential of promastigote forms by TMBP. TMBP concentrations of 0.86 μM (IC 50 ) and 1.72 μM (twice the IC 50 value) were used in this and subsequent experiments. Treatment decreased the intensity of total fluorescence of TMRE (0.86 and 1.72 μM) compared to the control group, indicating loss of integrity of the mitochondria (Fig. 4A). This behavior was also observed for the depolarizing agent CCCP. These findings suggest that TMBP inhibits the viability of L. amazonensis promastigotes by affecting the parasite's mitochondrial functions.
We also evaluated the production of ROS in the promastigote forms treated with TMBP. The treated parasites demonstrated an increase in ROS levels compared to the control group at both analyzed TMBP concentrations; 0.86 and 1.72 μM (Fig. 4B). The same was observed for the positive control, H 2 O 2 . ROS can act directly on Leishmania spp., causing the oxidation of proteins, lipids, and nucleic acids 38 , thus causing damage to the mitochondria that leads to their depolarization 39 . These results demonstrated that the leishmanicidal effect of TMBP on the promastigote forms of L. amazonensis was associated with the production of ROS and mitochondrial dysfunction, causing the parasite's death.
The effect of TMBP on the amastigote forms of L. amazonensis. To investigate the effect of TMBP on the amastigote forms of L. amazonensis, we first evaluated its cytotoxic action on two mammalian primary cells: murine macrophages and sheep erythrocytes. Concentrations of 0.03 to 15.60 µM did not alter the viability of peritoneal macrophages from BALB/c mice (Fig. 5A), presenting a CC 50 value of 53.93 µM. The selectivity index of TMBP was determined and revealed that this compound was 62.7 times more selective for parasites than the host cells (BALB/c mice). The results of hemolytic activity (Fig. 5B), expressed as a percentage of viability, indicated that the tested concentrations of TMBP did not cause hemolysis of sheep erythrocytes. These observations agree with the findings reported by our group that TMBP did not cause lysis in sheep red blood cells and also did not result in the loss of viability of murine peritoneal macrophages, thereby reinforcing its potential use as a drug 26 .
Considering that TMBP reduced the proliferation of promastigote forms and was not toxic to macrophages, we decided to investigate its effect against the intracellular amastigote forms of L. amazonensis. Despite the results showing no statistically significant decrease in the percentage of infected cells (Fig. 6A), there was a reduction www.nature.com/scientificreports/ in the number of intracellular parasites per macrophage (Fig. 6B). Furthermore, the two TMBP concentrations evaluated significantly decreased the mean number of amastigotes per macrophage by 35.2 and 46.2% (p ≤ 0.001 and p ≤ 0.0001, respectively) compared to the control. Macrophages are the primary host cells for Leishmania sp. 40 , and the intracellular localization of parasites represents an additional challenge for the treatment of this parasitosis since drugs must penetrate different cell layers, such as the cell membrane and the phagolysosome membrane to target the parasites specifically 41 . Thus, according to DNDi (Drugs for Neglected Diseases initiative), the ability to act on intracellular forms is required for potential antileishmanial drugs 42 .
In summary, TMBP acted on the intracellular parasite, reducing the number of viable amastigote forms in macrophages, allowing this compound to diffuse through the cell membrane, acting directly on the target without causing considerable toxic effects to the host cell. TMBP also did not demonstrate cytotoxic effects on peritoneal macrophages nor lysed sheep erythrocytes. Besides, cell death mechanisms resulted from the formation of reactive oxygen species and mitochondrial depolarization.  www.nature.com/scientificreports/ Molecular docking studies. Leishmania species express abundant surface protein antigens in promastigotes. Gp63 promotes the parasite's interaction with the host's defensive systems in this context. Another relevant target is the enzyme TR, which is involved in thiol-redox homeostasis and acts in the parasite's defense against oxidative agents. Since our results demonstrated that TMBP could induce ROS production, decrease cell size, and cause damage to mitochondria, we decided to perform molecular docking studies to obtain insights into the putative effects of TMBP on these targets. The consensus docking between at least two scoring functions that present the lowest value of RMSD (and < 2.0) for the identification of the pose and the intermolecular interactions on the binding site of LaGp63 were considered (Table 1) and presented a Fitness Score of 53.52.
The docking complex between the TMBP ligand and LaGp3 revealed that this biphenyl compound could interact with the enzyme by hydrophobic interactions involving amino acid residues: Val135, Val189, Ser218, Glu219, Val222, Leu223, Ala224, Trp225, Ala226, Val260, His267, Tyr327, Ser331, His332, and Pro344. Also, we observed hydrogen bonding at the active site, through hydroxyl groups of TBMP interacting with catalytic amino acids His263 and Glu264, in addition to metal interaction with the Zn 2+ ion (Fig. 7). Furthermore, Glu264 is reported to assist the position of the catalytic residues His263, His267, and His332, allowing the coordination with a zinc ion. In this way, these observed interactions might cause the indirect inactivation of LaGp63.
The clustering analysis of the docking simulation complexes between the ligand and LaTR showed that these compounds could interact with this enzyme by the hydrogen bonds present (Fitness Score = 46.22). TMBP accomplishes two relevant H-bond interactions with Arg280 and Gly279, in addition to hydrophobic interactions with Tyr191, Ile192, Val325, Ser323, Met326, Leu327, Cys357 (Fig. 8). In general, this possible affinity binding of TMBP at the active site and proximity to the enzyme's cofactor, FAD, might interfere with the TR enzyme's indirect inactivation of catalytic activity.
Combined analysis of both target enzymes (LaTR and LaGp63) suggests that the methoxyl and mainly the hydroxyl group of TMBP participated in all hydrogen bond interactions with crucial amino acid residues of both enzymes, with TMBP having a greater affinity for the LaGp63 target. In addition, the presence of the aromatic ring in the structure of TMBP increases its lipophilicity (cLogP = 2.44), facilitating the permeability of its entry into cell membranes of the promastigote or amastigote forms, where these enzymes can be found. Consequently, these results suggest that this biphenyl compound should be further studied for its antileishmanial activity.   www.nature.com/scientificreports/ by the National Academy of Sciences, USA. The experiment involving animals follows the recommendations described in the ARRIVE guidelines.
Determination of the cell size of the parasites. L. amazonensis promastigotes forms (10 6 cells/mL) were treated with TMBP (0.03; 0.24; 0.49; 0.98; 1.95; 3.9; 7.8, and 15.6 µM) and incubated for 24 h at 24 °C. Afterward, the promastigotes were collected and washed with PBS, then analyzed by flow cytometry using a BD Accuri™ C6 Plus flow cytometer. The forward scatter-heights (FSC-H) represent the cell sizes. A total of 10,000 events were acquired in the region corresponding to the parasites 44 .
Determination of the IC 50 , CC 50, and Selectivity Index (SI). The concentration of TMBP capable of inhibiting 50% of promastigote forms in culture (IC 50 ) and the concentration responsible for causing the death of 50% of peritoneal macrophages (CC 50 ) was calculated by non-linear regression using GraphPad software (Inc., USA, 5.00) from the data obtained in Sects. 2.9 and 2.14, respectively. In addition, the values corresponding to the IC 50 and twice this concentration (2 × IC 50 ) were used in the following experiments to determine the mechanism of action of TMBP on the promastigote forms. The selectivity index (SI) of TMBP was expressed 45 as: Viability analysis of peritoneal macrophages. The evaluation of the cytotoxic effects of TMBP on peritoneal macrophages was carried out according to the assay procedure of Mosmann (1983) 47 , based on the mitochondrial oxidation of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich www.nature.com/scientificreports/ Anti-amastigote assay. Peritoneal cells of BALB/c mice (5 × 10 5 cells/mL) were cultured in 24-well plates as previously described by Gonçalves et al. (2018). After infection, non-internalized promastigotes were removed by washing with PBS, and the adherent cells treated with TMBP at IC 50

Conclusion
For the first time, this study has identified the promising anti-leishmania potential of the biphenyl TMBP against promastigote forms of L. amazonensis as it showed high activity (IC 50 = 0.48 at 0.86 μM) in the shortest time of treatment, 24 h, displaying dose-dependent behavior. In addition, at the tested concentrations of TMBP, this biphenyl compound was 62.7 times more selective for promastigotes than the macrophage cells and did not cause hemolysis of sheep erythrocytes, indicating low cytotoxicity. TMBP-induced cell death in promastigotes was due to the generation of ROS, as well as mitochondrial dysfunction. TMBP was able to act on the intracellular parasite, reducing the number of viable amastigote forms within macrophages, suggesting that it can diffuse through the cell membrane, acting directly on the target, without causing considerable toxic effects on the host cell. Analysis of the consensual docking simulations on the LaTR and LaGp3 targets suggested that this biphenyl compound makes significant molecular interactions to inhibit these enzymes, with TMBP having a greater affinity for the LaGp63 target. The observed interactions occur nearby the Zn 2+ ion in the case of LaGp3, while for LaTR, the interactions are nearby the cofactor, FAD. These results demonstrated the crucial participation of the hydroxyl group of TMBP, as most important in binding interactions between the ligand and both enzyme targets, indicating a possible pharmacophoric group of this compound. www.nature.com/scientificreports/